Apoptosis is a form of cell death in animals that is executed by intrinsic cellular biochemical programs. Initiated by inter, and intracellular signals, apoptosis plays essential roles in animal development, maintaining normal tissue homeostasis, and elimination of tumorigenic and viral infected cells. Cancer and autoimmunity may arise when normal apoptosis is defective, resulting in excess numbers of cells. Conversely, apoptosis may be prematurely activated in diseases including neurodegenerative diseases, and neuron and cardiomyocyte death during stroke. Caspases are a group of intracellular proteases that carry out apoptosis. Caspases are synthesized as inactive zymogens that become activated when cells are committed to apoptosis. Caspase-3 is one of the major caspases that are responsible for generating many characteristic biochemical and morphological features of apoptosis. One of the major caspase activation pathways is initiated by mitochondria through the release of apoptotic protease activating factors such as cytochrome c and Smac. These factors trigger the activation of caspase-3 through a cascade of caspases. The cytochrome c-initiated caspase-3 activating pathway is negatively regulated by IAPs, another family of proteins that bind and inhibit caspase activities. Smac neutralizes IAP activity and ensure apoptosis to proceed when cells are damaged beyond repair. This grant proposes to continue the biochemical studies on the caspase-3 activating process focusing on the regulation by IAPs and Smac. The results generated should provide mechanistic insights into how a cell's sensitivity to a certain apoptotic stimulus is determined. Such knowledge should provide guidance to the diagnosis and therapy of the common human diseases listed above.